1. Field of the Invention
This invention is directed to methods for synthesizing very large collections of diverse pyrrolidine compounds on solid supports. This invention is further directed to methods for identifying and isolating pyrrolidine compounds with useful and diverse activities from such collections. This invention is still further directed to the incorporation of identification tags in such collections to facilitate identification of compounds with desired properties.
2. References
The following publications, patents and patent applications are cited in this application as superscript numbers:
.sup.1 Tsuge, et al., Recent Advances in Azomethine Ylide Chemistry, in "Advances in Heterocyclic Chemistry", Vol. 45, pp. 231-349, Academic Press, Inc. (1989) PA0 .sup.2 Cwirla, et al., Proc. Natl. Acad. Sci., USA, 87:6378-6382 (1990) PA0 .sup.3 Scott & Smith, Science, 249:386-390 (1990) PA0 .sup.4 Devlin, et al., Science, 249:404-406 (1990) PA0 .sup.5 Cull, et al., Proc. Natl. Acad. Sci., USA, 89:1865-1869 (1992) PA0 .sup.6 International Patent Application Publication No. WO 91/17271 PA0 .sup.7 International Patent Application Publication No. WO 91/19818 PA0 .sup.8 International Patent Application Publication No. WO 93/08278 PA0 .sup.9 International Patent Application Publication No. WO 91/05058 PA0 .sup.10 International Patent Application Publication No. WO 92/02536 PA0 .sup.11 International Patent Application Publication No. WO 93/06121 PA0 .sup.12 U.S. patent application Ser. No. 07/946,239 PA0 .sup.13 U.S. Pat. No. 5,143,854, issued Sep. 1, 1992 PA0 .sup.14 Adrill, et al., Tetrahedron, 44(15):4953-4966 (1988) PA0 .sup.15 Dorrity, et al., Tetrahedron, 44(15):4941-4952 (1988) PA0 .sup.16 Grigg, et al., Tetrahedron, 45(6):1723-1746 (1989) PA0 .sup.17 Grigg, Chem. Soc. Rev., 16:89-121 (1987) PA0 .sup.18 Barr, et al, Tetrahedron Letters, 30(35):4727-4730 (1989) PA0 .sup.19 Allway, et al., Tetrahedron Letters, 32(41):5817-5820 (1991) PA0 .sup.20 Barr, et al., Tetrahedron Letters, 31(45):6569-6572 (1990) PA0 .sup.21 Grigg, et al., Tetrahedron Letters, 21:2461-2464 (1980) PA0 .sup.22 Barr, et al., Tetrahedron, 44(2):557-570 (1988) PA0 .sup.23 Amornraksa, et at., Tetrahedron, 45:(14):4649-4668 (1989) PA0 .sup.24 Armstrong, et al., Tetrahedron, 41:(17):3547-3558 (1985) PA0 .sup.25 Grigg, et al., Tetrahedron, 49(38):8679-8690 (1993) PA0 .sup.26 Grigg, et al., Tetrahedron, 48(47):10431-10442 (1992) PA0 .sup.27 Grigg, et al., Tetrahedron, 43(24):5887-5898 (1987) PA0 .sup.28 Grigg, et al., J. Chem. Soc. Chem. Commun., pp. 47-51 (1987) PA0 .sup.29 Grigg, et al., Tetrahedron, 48(47):10423-10430 (1992) PA0 .sup.30 Grigg, et al., Tetrahedron Letters, 24(41):4457-4460 (1983) PA0 .sup.31 Aly, et al., Tetrahedron, 50(3):895-906 (1994)
All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art
Ligands for macromolecular receptors can be identified by screening diverse collections of compounds, e.g., peptides, produced through either molecular biological or synthetic chemical techniques. For example, recombinant peptide libraries have been generated by inserting degenerate oligonucleotides into genes encoding capsid proteins of filamentous bacteriophage and the DNA-binding protein Lac I..sup.2-8 These random libraries contain more than 10.sup.9 different peptides, each fused to a larger protein sequence that is physically linked to the genetic material encoding it. Such libraries are efficiently screened for interaction with a receptor by several rounds of affinity purification, the selected exposition or display vectors being amplified in E. coli and the DNA of individual clones sequenced to reveal the identity of the peptide responsible for receptor binding..sup.9,10
Other disclosed methods for screening libraries of compounds for binding properties to a receptor include methods wherein each member of the library is tagged with a unique identifier tag to facilitate identification of compounds having binding properties.sup.11,23 or where the library comprises a plurality of compounds synthesized at specific locations on the surface of a solid substrate wherein the receptor is appropriately labeled to identify binding, e.g., fluorescent or radioactive labels. Correlation of the labelled receptor bound to the substrate with its location on the substrate identifies the binding ligand..sup.13
Central to these methods is the screening of a multiplicity of compounds in the library and the ability to identify the structures of the compounds which have a requisite binding affinity for the receptor. Preferably, in order to facilitate synthesis and identification, the compounds in the library are typically formed on solid supports wherein the compound is covalently attached to the support via a cleavable or non-cleavable linking arm. In this regard, the diversity of naturally occurring amino acids permits the generation of extensive peptide libraries on such solid supports without resort to the use of synthetic amino acids which can include derivatives of naturally occurring amino acids. These libraries are then screened to identify "lead compounds" having good binding affinity to the receptor.
Pharmaceutical drug discovery relies heavily on studies of structure-activity relationships wherein the structure of "lead compounds" is typically altered to determine the effect of the alteration on activity. When the lead compound comprises one or more amino acids (e.g., a peptide), alteration of the structure of one or more of the amino acid(s) permits evaluation of the effect of the structural alteration on activity. Thus libraries of compounds derived from a lead compound can be created by including derivatives of the amino acids in the peptides and repeating the screening procedures.
The use of such amino acid derivatives in these libraries has been disclosed in the art..sup.12,13 Ideally, the amino acid derivative is synthesized in situ on the solid support so that the support can be tagged to identify the synthetic steps employed and/or the derivative incorporated onto the support. However, relatively simple synthetic methods to produce a diverse collection of such derivatives on the supports are often not available.
One particular class of compounds which would be useful for inclusion in screening libraries are pyrrolidine compounds, including proline and derivatives thereof. These compounds form the basis of an important class of compounds having diverse pharmaceutical and chemical properties. Proline, itself, is often included in the structure of peptides having receptor binding activity and derivatives of proline form important pharmaceutical compositions such as Captopril, a commercial anti-hypertensive agent. Similarly, pyrrolidine compounds are the central skeletal feature on numerous alkaloids..sup.1
The inclusion of certain proline derivatives into such libraries is well known in the art. However, a simple procedure for the in situ incorporation of a multiplicity of pyrrolidine derivatives on solid supports is not previously known. The ability to synthesize a multiplicity of pyrrolidine derivatives on a solid support or on different solid supports would enhance the structural variation of a library and provide important structure-activity information.